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FIG 18.1 Phosphoinositide metabolism and the structure of PI(3,4,5)P3.
A reaction scheme outlining the pathways of formation of the seven principal phosphoinositides. Selected kinases are shown in red. PTEN and SHIP are phosphatases. The PI 3-kinases phosphorylate the inositol headgroup at the 3 position. Other kinases include the phosphatidylinositol phosphate kinases (PIPKins) of which PI(4)P 5-kinase is a PIPKinI and PIKfyve a PIPKinIII.
this scheme may seem complicated, it is important to bear in mind that the kinases are restricted to particular membranes so that the different phospholipids are enriched at specific locations. Thus PI(4)P is the predominant phosphoinositide on cytosolic surface of the Golgi apparatus,
while PI(3)P, PI(4)P and PI(3,5)P2 are enriched on the surfaces of the vesicular compartments that characterize the endocytic pathway, such as endosomes. PI(4,5)P2 and PI(3,4)P2 are predominant on the plasma membrane. The head groups of each of these phosphoinositides then provide specific tethers for the relevant proteins that recruit the machinery of endocytosis, exocytosis and intracellular vesicular transport (see also Chapter 24).
PI 3-kinase, PI(3,4)P2 and PI(3,4,5)P3
While PI(4)P and PI(4,5)P2 are present in resting cells, the 3-phosphoinositides PI(3,4)P2 and PI(3,4,5)P3 are only formed upon the activation of PI 3-kinase
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(Figure 18.1). PI(3,4,5)P3 was first detected in human neutrophils after stimulation with formylmethionyl peptides.12 It was initially thought to be another substrate for PLC, and thus the source of a water-soluble second messenger (inositol-1,3,4,5-tetrakisphosphate or IP4), but it soon became clear that the lipid itself is a signalling entity.
Phosphatidylinositol occupies a special place among phospholipids, because its head group offers a multiplicity of phosphorylation sites. Although inositol lipids have been detected in analyses of bacteria, there is no indication that any of them plays a regulatory role. However, it is clear that some bacteria rely on the inositide metabolism of host cells to regulate their invasiveness.13 PI is present in all eukaryotic cells, though its metabolism in unicellular organisms such as yeast is restricted, since they lack the means to generate either PI(4)P or PI(4,5)P2. The substrate specificity of the yeast PI 3-kinase (coded by the gene VPS34) is appropriately limited to PI and therefore it only generates
the monophosphorylated derivative PI(3)P (which can then be converted to PI(3,5)P2, essential for Golgi membrane recycling14,15). The PI 3-kinases catalysing the formation of PI(3,4)P2 and PI(3,4,5)P3 probably evolved with the need for more complex forms of metabolic regulation following the emergence of the metazoans.
Inositol phospholipids having a phosphate group at the 3 position do not serve directly as substrates for phospholipase C. Instead, they are metabolized by the hydrolysis of the phosphate groups at the 3 - and 5 - positions by the phosphatases PTEN and SHIP respectively (Figure 18.1). Both the 3-kinases and the 3-phosphatases are regulated by receptor-mediated processes.
A family of PI 3-kinases
Cloning and screening strategies have revealed a family of PI 3-kinases consisting of three types that have distinct substrates and various forms of regulation (Figure 18.2). They all have four homologous regions, the kinase domain being the most conserved16.
Type I PI 3-kinases
Type I PI 3-kinases phosphorylate PI, PI(4)P and PI(4,5)P2 (the preferred substrate). They are heterodimers in vivo comprising a regulatory (p55 or p85) subunit that maintains the enzyme in a low activity state and a catalytic subunit (p110). Each exists in various forms (Figure 18.2). Their multi-domain structure (in particular p85) enables them to interact with other signalling proteins allowing activation.17 The SH2 domains enable them to bind to phosphotyrosine residues. Similarly, the SH3 domains allow interaction with proline-rich sequences, present for instance in the focal adhesion kinase FAK, the adaptor molecule Shc, the GTPase-activating protein Cdc42GAP, or the regulator of TCR signalling, Cbl.18 In addition, the p85-subunit contains a
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FIG 18.2 Classification of phosphoinositide 3-kinases.
This is based on the structure of the kinase domain and there are three main types. Type I enzymes are heterodimeric and are subdivided into two groups. Group A comprises either p110 , - , or - with the regulatory subunit p85 (or its splice variants p55 and p50 ) or p55 . Group B has one member, p110 associated with the p101 regulatory subunit. This is involved in G -mediated activation of the kinase. Type II comprises three isoforms, C2 , - , and - , none of which requires a regulatory subunit for its activity. Type III has only one member, PI 3-kinase-III (also known as Vsp34-like) which has a p150 regulatory subunit. The mechanisms regulating the type II and III kinases are not yet established.
BCR/GAP homology domain19 that interacts with Rac and Cdc42, members of the Rho family of GTPases, providing yet further opportunities for regulation20 (Figure 18.4).
There are four isoforms of the p110 catalytic-subunit ( , , , and ) all of which contain a kinase domain and a Ras binding site.21 In addition, the ,, and isoforms possess interaction sites for the p85-subunit. The type I
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enzymes can be further subdivided. Type IA enzymes ( , , and ) interact through their SH2 domains with phosphotyrosines present on either protein tyrosine kinases or to docking proteins such as insulin receptor substrates (IRS, see below) or LAT (see page 517). Uniquely, the class IA enzymes activate protein kinase B (PKB, also called Akt, see below). Type IB consists of a single member, p110 linked to a unique regulatory subunit, p101 , that has no apparent sequence homology with other proteins. Importantly, p110 can be regulated by -subunits.22,23
The type I PI 3-kinase enzymes respond to different upstream signals and have different functions. This is apparent in macrophages in which distinct isotypes modulate separate cellular responses. Here, mitogenic signalling induced by colony stimulating factor-1 is mediated by p110 , whereas actin organization and cell migration require the or isoforms.24
In mammalian cells the class I PI 3-kinases have roles in the modification of intracellular membranes, affecting the recruitment of the fission and fusion machinery necessary for intracellular vesicle traffic. The class I PI 3-kinases are also involved in the formation of phagosomes.25
Type II PI 3-kinases
The three members of this group, , - , and - , have substrate specificity for PI and PI(4)P. They are all monomeric and possess a C-terminal C2 domain. Their mode of activation is unclear.
Type III PI 3-kinases
These are represented by the human homologues of the yeast gene product VPS34. They only phosphorylate PI to form PI(3)P. Like the yeast enzyme, the human form is tightly coupled to a regulatory subunit (p150, homologue of VPS15), an serine/threonine kinase which both phosphorylates and recruits the catalytic unit to membranes. Under conditions of starvation, the activity of hVps34 is inhibited, causing restriction of the synthesis of enzymes involved in a pathway that acts via the kinase complex mTOR/Raptor (see below). How hVps34 regulates mTOR/Raptor activity is not known, but it is clear that it is a key component of the nutrient sensing system. In yeast, hVsp34 is involved in autophagy, a rescue mechanism that allows renewal of cellular components in times of prolonged starvation.26
Studying the role of PI 3-kinase
Wortmannin, an inhibitor of PI 3-kinase
It might be thought that the availability of an inhibitor of the PI 3-kinases, in this case wortmannin, would have provided an unambiguous key to the understanding of the pathways and cellular functions that they control. This antifungal antibiotic, isolated from Penicillium wortmannii, was originally
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